Affiliations: 1: Institute of Structural Molecular Biology, Birkbeck College and University College London, London, United Kingdom;
2: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri;
3: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri

Beauty in asymmetry: the sporulation transcription factor σF bound to the anti-σ factor SpoIIAB. (A) The domain of B. stearothermophilus σF (σ3F) visible in the crystal structure is shown as a molecular surface and colored blue. The surface of σ3F that interacts with core RNAP is colored gray. The SpoIIAB dimer, drawn in ribbons and colored magenta, extensively interacts with the core-binding surface of σ3F. The nucleotide, ADP in this structure, is drawn in stick form and sits in the serine kinase domain of the anti-σ factor. (B) This view is flipped 180° from that in panel A to show the asymmetric interaction of σ3F and the SpoIIAB dimer. SpoIIAB and σ3F are both drawn as molecular surfaces and colored as in panel A. Residues of SpoIIAB that are known to be critical for displacement and interaction with the anti-anti-σ factor SpoIIAA are colored yellow on SpoIIAB. The asymmetry of the interaction led to a docking model, which predicted that SpoIIAA displaces σF by docking onto the SpoIIAB monomer labeled AB2, where Arg20, a residue critical for displacement, was exposed (Campbell et al., 2002a; Ho et al., 2003). The docking of SpoIIAA would occur via interactions with Glu104, the ATP-binding pocket (shown in grey and represented by Thr 49), and Arg20.

10.1128/9781555818395/plate-2_thmb.gif

10.1128/9781555818395/plate-2.gif

Click to view

Color Plate 2 (chapter 1).

Beauty in asymmetry: the sporulation transcription factor σF bound to the anti-σ factor SpoIIAB. (A) The domain of B. stearothermophilus σF (σ3F) visible in the crystal structure is shown as a molecular surface and colored blue. The surface of σ3F that interacts with core RNAP is colored gray. The SpoIIAB dimer, drawn in ribbons and colored magenta, extensively interacts with the core-binding surface of σ3F. The nucleotide, ADP in this structure, is drawn in stick form and sits in the serine kinase domain of the anti-σ factor. (B) This view is flipped 180° from that in panel A to show the asymmetric interaction of σ3F and the SpoIIAB dimer. SpoIIAB and σ3F are both drawn as molecular surfaces and colored as in panel A. Residues of SpoIIAB that are known to be critical for displacement and interaction with the anti-anti-σ factor SpoIIAA are colored yellow on SpoIIAB. The asymmetry of the interaction led to a docking model, which predicted that SpoIIAA displaces σF by docking onto the SpoIIAB monomer labeled AB2, where Arg20, a residue critical for displacement, was exposed (Campbell et al., 2002a; Ho et al., 2003). The docking of SpoIIAA would occur via interactions with Glu104, the ATP-binding pocket (shown in grey and represented by Thr 49), and Arg20.

The anti-σ wrap: structure of the periplasmic stress response ECF σ factor, σE, with the cytoplasmic domain of its anti-σ RseA. (A) Ribbon diagram of E. coli σE with the anti-σ domain of RseA. Domains of σE are colored as follows: σE2, green;σE4, yellow; σE2-σE4 linker, red; RseA (N-terminal 66 residues), magenta (shown as a coil). The anti-σ domain is sandwiched between σE2 and σE4. (B) Surface representation of σE, color coded as in panel A but with the core-binding surfaces shaded grey on σE2 and σE4. RseA, which extensively occludes the core-interacting surfaces of σE, is shown as a magenta ribbon.

10.1128/9781555818395/plate-3_thmb.gif

10.1128/9781555818395/plate-3.gif

Click to view

Color Plate 3 (chapter 1).

The anti-σ wrap: structure of the periplasmic stress response ECF σ factor, σE, with the cytoplasmic domain of its anti-σ RseA. (A) Ribbon diagram of E. coli σE with the anti-σ domain of RseA. Domains of σE are colored as follows: σE2, green;σE4, yellow; σE2-σE4 linker, red; RseA (N-terminal 66 residues), magenta (shown as a coil). The anti-σ domain is sandwiched between σE2 and σE4. (B) Surface representation of σE, color coded as in panel A but with the core-binding surfaces shaded grey on σE2 and σE4. RseA, which extensively occludes the core-interacting surfaces of σE, is shown as a magenta ribbon.

All tied up: structure of the flagellar σFliA with its anti-σ FlgM. (A) σFliA is depicted as a ribbon structure with the domains colored as follows: σFliA2, green; σFliA3, blue; σFliA3-σFliA4 linker, red; σFliA4, yellow. FlgM, shown as a magenta coil, threads around the compacted σ factor, interacting extensively with σFliA2 and σFliA4. (B) Same view as shown in panel A, with σFliA shown as molecular surface and FlgM shown as a ribbon. Colors are the same as in panel A, except that the surfaces of σFliA that interact with core RNAP are shaded grey. FlgM effectively occludes the core-binding interfaces on both σFliA2 and σFliA4.

10.1128/9781555818395/plate-4_thmb.gif

10.1128/9781555818395/plate-4.gif

Click to view

Color Plate 4 (chapter 1).

All tied up: structure of the flagellar σFliA with its anti-σ FlgM. (A) σFliA is depicted as a ribbon structure with the domains colored as follows: σFliA2, green; σFliA3, blue; σFliA3-σFliA4 linker, red; σFliA4, yellow. FlgM, shown as a magenta coil, threads around the compacted σ factor, interacting extensively with σFliA2 and σFliA4. (B) Same view as shown in panel A, with σFliA shown as molecular surface and FlgM shown as a ribbon. Colors are the same as in panel A, except that the surfaces of σFliA that interact with core RNAP are shaded grey. FlgM effectively occludes the core-binding interfaces on both σFliA2 and σFliA4.

Conserved domains of two-component pathways. (A) The simplest of the two-component pathways, a phosphotransfer pathway, consists of a single-step transfer of a phosphoryl group between a His of an HK and an Asp of a downstream RR. A typical HK contains a variable periplasmic sensing domain (gray) and a conserved kinase core consisting of a dimerization domain (blue) and a catalytic ATP-binding domain (yellow). The RR contains a conserved N-terminal regulatory domain (green) and a variable Cterminal effector domain (gray). (B) Two-component pathways can also be more complex, involving components that contain multiple phosphodonor and acceptor sites. These phosphorelay systems typically involve hybrid kinases that contain a histidine kinase core together with an RR regulatory domain, a separate His-containing phosphotransfer domain, and a separate RR. However, the conserved domains are modular and are found in a variety of different arrangements in various phosphorelay pathways. (C to E) Ribbon images are shown for representative conserved domains: the His-containing dimerization domain (blue) with the His residue that is phosphorylated depicted in ball-and-stick representation (PDB code 1JOY) (C), the catalytic ATP-binding domain (yellow) with the bound nucleotide shown in ball-and-stick representation (PDB code 1I5D) (D), and the Asp-containing regulatory domain (green) with the Asp residue that is phosphorylated depicted in ball-and-stick representation (PDB code 2CHE) (E).

10.1128/9781555818395/plate-5_thmb.gif

10.1128/9781555818395/plate-5.gif

Click to view

Color Plate 5 (chapter 2).

Conserved domains of two-component pathways. (A) The simplest of the two-component pathways, a phosphotransfer pathway, consists of a single-step transfer of a phosphoryl group between a His of an HK and an Asp of a downstream RR. A typical HK contains a variable periplasmic sensing domain (gray) and a conserved kinase core consisting of a dimerization domain (blue) and a catalytic ATP-binding domain (yellow). The RR contains a conserved N-terminal regulatory domain (green) and a variable Cterminal effector domain (gray). (B) Two-component pathways can also be more complex, involving components that contain multiple phosphodonor and acceptor sites. These phosphorelay systems typically involve hybrid kinases that contain a histidine kinase core together with an RR regulatory domain, a separate His-containing phosphotransfer domain, and a separate RR. However, the conserved domains are modular and are found in a variety of different arrangements in various phosphorelay pathways. (C to E) Ribbon images are shown for representative conserved domains: the His-containing dimerization domain (blue) with the His residue that is phosphorylated depicted in ball-and-stick representation (PDB code 1JOY) (C), the catalytic ATP-binding domain (yellow) with the bound nucleotide shown in ball-and-stick representation (PDB code 1I5D) (D), and the Asp-containing regulatory domain (green) with the Asp residue that is phosphorylated depicted in ball-and-stick representation (PDB code 2CHE) (E).

ATP-binding domain of chemotaxis HK CheA. (A) The crystal structure of the nucleotide-binding domain of CheA (residues 354-542; PDB code 1B3Q) is shown as a ribbon representation, with labels designating the highly conserved motifs (cyan) that surround the nucleotide-binding site: the N box, the G1 box, the F box, and the G2 box. The F-box and G-box motifs lie within a disordered region known as the ATP lid (magenta). (B) In the crystal structure of CheA with ADPCP-Mg2+ bound (PDB code 1I58), the ATP lid (magenta) adopts a more ordered, closed conformation relative to the poorly defined corresponding region present in the structure of CheA without bound nucleotide. The ATP analog is shown in ball-and-stick representation.

10.1128/9781555818395/plate-6_thmb.gif

10.1128/9781555818395/plate-6.gif

Click to view

Color Plate 6 (chapter 2).

ATP-binding domain of chemotaxis HK CheA. (A) The crystal structure of the nucleotide-binding domain of CheA (residues 354-542; PDB code 1B3Q) is shown as a ribbon representation, with labels designating the highly conserved motifs (cyan) that surround the nucleotide-binding site: the N box, the G1 box, the F box, and the G2 box. The F-box and G-box motifs lie within a disordered region known as the ATP lid (magenta). (B) In the crystal structure of CheA with ADPCP-Mg2+ bound (PDB code 1I58), the ATP lid (magenta) adopts a more ordered, closed conformation relative to the poorly defined corresponding region present in the structure of CheA without bound nucleotide. The ATP analog is shown in ball-and-stick representation.

His-containing phosphotransfer (HPt) domains. HPt domains are represented by Saccharomyces cerevisiae YPD1 (PDB code 1C02) (A) and B. subtilis Spo0B (PDB code 1IXM) (B). The HPt proteins contain a four-helix bundle (blue) that can be composed of either one chain with one active His residue (red) per structural unit, as in YPD1, or two chains with two active His residues per structural unit, as in the Spo0B dimer. Additional structural elements (brown) are variable among HPt proteins.

10.1128/9781555818395/plate-7_thmb.gif

10.1128/9781555818395/plate-7.gif

Click to view

Color Plate 7 (chapter 2).

His-containing phosphotransfer (HPt) domains. HPt domains are represented by Saccharomyces cerevisiae YPD1 (PDB code 1C02) (A) and B. subtilis Spo0B (PDB code 1IXM) (B). The HPt proteins contain a four-helix bundle (blue) that can be composed of either one chain with one active His residue (red) per structural unit, as in YPD1, or two chains with two active His residues per structural unit, as in the Spo0B dimer. Additional structural elements (brown) are variable among HPt proteins.

Conserved activation mechanism of the RR regulatory domain. Side chains of the conserved Asp, Ser/Thr, and Phe/Tyr are displayed in conformations observed in unphosphorylated (red) and phosphorylated (blue) RRs on a representative backbone (green, with α-helices labeled). In RRs phosphorylated at the conserved Asp, the side chain of the Ser/Thr is reoriented to form a hydrogen bond with a phosphate oxygen. The side chain of Phe/Tyr is positioned inward, toward the site of phosphorylation, filling the cavity vacated by the rotated Ser/Thr. The repositioning of these residues is involved in the propagation of a subtle conformational change to the α4-β5-α5 face of the regulatory domain.

10.1128/9781555818395/plate-8_thmb.gif

10.1128/9781555818395/plate-8.gif

Click to view

Color Plate 8 (chapter 2).

Conserved activation mechanism of the RR regulatory domain. Side chains of the conserved Asp, Ser/Thr, and Phe/Tyr are displayed in conformations observed in unphosphorylated (red) and phosphorylated (blue) RRs on a representative backbone (green, with α-helices labeled). In RRs phosphorylated at the conserved Asp, the side chain of the Ser/Thr is reoriented to form a hydrogen bond with a phosphate oxygen. The side chain of Phe/Tyr is positioned inward, toward the site of phosphorylation, filling the cavity vacated by the rotated Ser/Thr. The repositioning of these residues is involved in the propagation of a subtle conformational change to the α4-β5-α5 face of the regulatory domain.

Structures of full-length multidomain RRs. Ribbon representations of Salmonella enterica serovar Typhimurium methylesterase CheB (PDB code 1A2O) (A), E. coli transcription factor NarL (PDB code 1A04) (B), T. maritima transcription factor DrrB (PDB code 1P2F) (C) T. maritima transcription factor DrrD (PDB code 1KGS) (D) are aligned with similar orientations of the regulatory domain. The regulatory domain (green) is highly conserved among RRs, and all contain a conserved Asp (red) as the site of phosphorylation. The effector domain (cyan) of the methylesterase CheB contains a catalytic triad (magenta) at its active site. The transcription factor effector domains (cyan) of NarL, DrrB, and DrrD contain a recognition helix (magenta) that binds DNA. The regulatory and effector domains are joined by linkers (gray), which are often disordered in the crystal structures (dashed lines). The interdomain interface varies in size and involves different subsets of the α4-β5-α5 face of the regulatory domain, implying the presence of different mechanisms of intramolecular communication in different RRs.

10.1128/9781555818395/plate-9_thmb.gif

10.1128/9781555818395/plate-9.gif

Click to view

Color Plate 9 (chapter 2).

Structures of full-length multidomain RRs. Ribbon representations of Salmonella enterica serovar Typhimurium methylesterase CheB (PDB code 1A2O) (A), E. coli transcription factor NarL (PDB code 1A04) (B), T. maritima transcription factor DrrB (PDB code 1P2F) (C) T. maritima transcription factor DrrD (PDB code 1KGS) (D) are aligned with similar orientations of the regulatory domain. The regulatory domain (green) is highly conserved among RRs, and all contain a conserved Asp (red) as the site of phosphorylation. The effector domain (cyan) of the methylesterase CheB contains a catalytic triad (magenta) at its active site. The transcription factor effector domains (cyan) of NarL, DrrB, and DrrD contain a recognition helix (magenta) that binds DNA. The regulatory and effector domains are joined by linkers (gray), which are often disordered in the crystal structures (dashed lines). The interdomain interface varies in size and involves different subsets of the α4-β5-α5 face of the regulatory domain, implying the presence of different mechanisms of intramolecular communication in different RRs.

Ribbon representations of chemotaxis proteins. (A) CheW (PDB code 1K0S) couples CheA kinase to the chemotaxis receptors. (B) The P1 domain of CheA (coordinates provided by F. W. Dahlquist) is an HPt-like domain containing a four-helix bundle (blue) and an additional helix (brown) that contains the site of His phosphorylation (red). (C) The P2 domain of CheA (PDB code 1FWP) serves as an interaction site for the RRs CheY and CheB (structures shown in Color Plates 5E and 9A, respectively). The helices serve as an interaction site for the α4-β5-α5 face of CheY. (D) The dimerization domain (blue), catalytic kinase domain (yellow), and CheW interaction/coupling domain (orange) comprise the C-terminal half of CheA (PDB code 1B3Q). To date, this is the only structure of an intact kinase core. (E) The phosphatase CheZ (purple) accelerates the dephosphorylation of the RR CheY (green) in some species of bacteria. In the CheY-CheZ complex (PDB code 1KMI), two monomers of CheZ dimerize to form a four-helix bundle. An additional C-terminal helix of CheZ that caps a long, disordered tail tethers CheY through interaction with the α4-β5-α5 face and facilitates an otherwise weak interaction between the active sites of CheY and CheZ. (F) The methyltransferase CheR (PDB code 1BC5) bound to the product of the methylation reaction, S-adenosylhomocysteine (shown in ball-and-stick representation) is shown bound to a peptide (red) that corresponds to a recognition sequence (NWETF) at the C termini of chemotaxis receptors. The pentapeptide-binding β-subdomain (magenta) is a specialized insertion into the methyltransferase fold, which serves to tether CheR to the chemotaxis receptors.

10.1128/9781555818395/plate-10_thmb.gif

10.1128/9781555818395/plate-10.gif

Click to view

Color Plate 10 (chapter 2).

Ribbon representations of chemotaxis proteins. (A) CheW (PDB code 1K0S) couples CheA kinase to the chemotaxis receptors. (B) The P1 domain of CheA (coordinates provided by F. W. Dahlquist) is an HPt-like domain containing a four-helix bundle (blue) and an additional helix (brown) that contains the site of His phosphorylation (red). (C) The P2 domain of CheA (PDB code 1FWP) serves as an interaction site for the RRs CheY and CheB (structures shown in Color Plates 5E and 9A, respectively). The helices serve as an interaction site for the α4-β5-α5 face of CheY. (D) The dimerization domain (blue), catalytic kinase domain (yellow), and CheW interaction/coupling domain (orange) comprise the C-terminal half of CheA (PDB code 1B3Q). To date, this is the only structure of an intact kinase core. (E) The phosphatase CheZ (purple) accelerates the dephosphorylation of the RR CheY (green) in some species of bacteria. In the CheY-CheZ complex (PDB code 1KMI), two monomers of CheZ dimerize to form a four-helix bundle. An additional C-terminal helix of CheZ that caps a long, disordered tail tethers CheY through interaction with the α4-β5-α5 face and facilitates an otherwise weak interaction between the active sites of CheY and CheZ. (F) The methyltransferase CheR (PDB code 1BC5) bound to the product of the methylation reaction, S-adenosylhomocysteine (shown in ball-and-stick representation) is shown bound to a peptide (red) that corresponds to a recognition sequence (NWETF) at the C termini of chemotaxis receptors. The pentapeptide-binding β-subdomain (magenta) is a specialized insertion into the methyltransferase fold, which serves to tether CheR to the chemotaxis receptors.

Structure of FimH. (A) Coordinates were taken from the crystal structure of the FimCH complex (1KLF). In this figure, the ribbon representation is of FimH only. The lectin-binding domain is shown in magenta, and the pilin domain is shown in blue. D-Mannose is located at the top of the molecule, where carbon atoms are yellow and oxygen atoms are red. (B) Molecular surface representation with electrostatic potential surface (Nicholls et al., 1991), with positively charged residues shown in blue, negatively charged residues shown in red, neutral and hydrophobic residues shown in white. Residues defining the hydrophobic ridge around the mannose-binding pocket are labeled. (C) Mannose-binding site, with FimH residues shown in magenta. Mannose residues are shown with carbon atoms in yellow and oxygen atoms in red. Panel B adapted from Hung et al. (2002) with permission.

10.1128/9781555818395/plate-11_thmb.gif

10.1128/9781555818395/plate-11.gif

Click to view

Color Plate 11 (chapter 3).

Structure of FimH. (A) Coordinates were taken from the crystal structure of the FimCH complex (1KLF). In this figure, the ribbon representation is of FimH only. The lectin-binding domain is shown in magenta, and the pilin domain is shown in blue. D-Mannose is located at the top of the molecule, where carbon atoms are yellow and oxygen atoms are red. (B) Molecular surface representation with electrostatic potential surface (Nicholls et al., 1991), with positively charged residues shown in blue, negatively charged residues shown in red, neutral and hydrophobic residues shown in white. Residues defining the hydrophobic ridge around the mannose-binding pocket are labeled. (C) Mannose-binding site, with FimH residues shown in magenta. Mannose residues are shown with carbon atoms in yellow and oxygen atoms in red. Panel B adapted from Hung et al. (2002) with permission.

Structure of PapGII. (A) The ribbon representation of the PapGII lectin-binding domain is shown in orange. The globoside receptor is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The PapGII-binding site is shown with PapGII residues in orange. The globoside polysaccharide is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

10.1128/9781555818395/plate-12_thmb.gif

10.1128/9781555818395/plate-12.gif

Click to view

Color Plate 12 (chapter 3).

Structure of PapGII. (A) The ribbon representation of the PapGII lectin-binding domain is shown in orange. The globoside receptor is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The PapGII-binding site is shown with PapGII residues in orange. The globoside polysaccharide is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

Structure of GafD. (A) The ribbon representation of the GafD lectin-binding domain is shown in green. The GlcNAc receptor is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The GafD-binding site is shown with GafD residues shown in green. The GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

10.1128/9781555818395/plate-14_thmb.gif

10.1128/9781555818395/plate-14.gif

Click to view

Color Plate 14 (chapter 3).

Structure of GafD. (A) The ribbon representation of the GafD lectin-binding domain is shown in green. The GlcNAc receptor is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The GafD-binding site is shown with GafD residues shown in green. The GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

Structure of F17a-G. (A) The ribbon representation of the lectin-binding domain of F17a-G is depicted in blue. GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The F17a-G-binding site with F17a-G residues shown in green. The GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

10.1128/9781555818395/plate-15_thmb.gif

10.1128/9781555818395/plate-15.gif

Click to view

Color Plate 15 (chapter 3).

Structure of F17a-G. (A) The ribbon representation of the lectin-binding domain of F17a-G is depicted in blue. GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (B) The F17a-G-binding site with F17a-G residues shown in green. The GlcNAc is shown with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

Variants of F17a-G. (A) The front side of the molecule containing the natural variants of the F17a-G lectin-binding domain is shown in green. (B) The back side of the molecule is depicted. GlcNAc is shown in the binding site, with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

10.1128/9781555818395/plate-16_thmb.gif

10.1128/9781555818395/plate-16.gif

Click to view

Color Plate 16 (chapter 3).

Variants of F17a-G. (A) The front side of the molecule containing the natural variants of the F17a-G lectin-binding domain is shown in green. (B) The back side of the molecule is depicted. GlcNAc is shown in the binding site, with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue.

Superposition of receptor-bound and apo-F17a-G binding sites. The receptor-bound structure is shown in magenta, while the apo structure is show in cyan. Solvent molecules are shown as magenta or cyan spheres.

10.1128/9781555818395/plate-17_thmb.gif

10.1128/9781555818395/plate-17.gif

Click to view

Color Plate 17 (chapter 3).

Superposition of receptor-bound and apo-F17a-G binding sites. The receptor-bound structure is shown in magenta, while the apo structure is show in cyan. Solvent molecules are shown as magenta or cyan spheres.

Structural alignment of the lectin-binding domains. Structural alignment was performed by using a three-dimensional protein structure comparison and alignment program (Shindyalov and Bourne, 1998). The lectin-binding domain is shown as a molecular surface, while the receptor is shown as sticks with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (A) FimH; (B) PapGII; (C) GafD; (D) F17a-G; (E) Fim rotated 90° to show the mannose-binding pocket.

10.1128/9781555818395/plate-18_thmb.gif

10.1128/9781555818395/plate-18.gif

Click to view

Color Plate 18 (chapter 3).

Structural alignment of the lectin-binding domains. Structural alignment was performed by using a three-dimensional protein structure comparison and alignment program (Shindyalov and Bourne, 1998). The lectin-binding domain is shown as a molecular surface, while the receptor is shown as sticks with carbon atoms in yellow, oxygen atoms in red, and nitrogen atoms in blue. (A) FimH; (B) PapGII; (C) GafD; (D) F17a-G; (E) Fim rotated 90° to show the mannose-binding pocket.

Structure of the EPEC Tir IBD in complex with intimin. (a) The crystal structure of the complex reveals an antiparallel Tir IBD-intimin dimer. The binding of Tir IBD is mediated by the C-terminal-most domain of intimin, D3. Tir IBD binding does not dramatically influence the structural integrity of intimin (the Cα positions of the isolated intimin structure can be superimposed onto that of the complex structure with a root mean squared deviation of 0.67 Å). (b) Close-up view of the Tir-binding pocket on intimin. Intimin binds specifically to the β-hairpin and the N terminus of helix HA of the Tir IBD dimer. The binding interface is primarily hydrophobic, but electrostatic interactions, such as the salt bridge between Lys927 of intimin and Glu312 of the Tir IBD, and backbone interactions also contribute to the specificity and affinity of the interaction. (c) Complementary electrostatics between Tir IBD and intimin. An electrostatic surface potential representation of the Tir IBD-intimin complex as computed by GRASP (Nicholls et al., 1991) is shown here, with red representing negatively charged regions and blue representing positively charged regions. The Tir IBD has an overall negative charge, but the dimerization interface is predominantly hydrophobic. Intimin has a complementary net positive charge with a positively charged tip pointing towards the α-helices of the Tir IBD dimer.

10.1128/9781555818395/plate-19_thmb.gif

10.1128/9781555818395/plate-19.gif

Click to view

Color Plate 19 (chapter 4).

Structure of the EPEC Tir IBD in complex with intimin. (a) The crystal structure of the complex reveals an antiparallel Tir IBD-intimin dimer. The binding of Tir IBD is mediated by the C-terminal-most domain of intimin, D3. Tir IBD binding does not dramatically influence the structural integrity of intimin (the Cα positions of the isolated intimin structure can be superimposed onto that of the complex structure with a root mean squared deviation of 0.67 Å). (b) Close-up view of the Tir-binding pocket on intimin. Intimin binds specifically to the β-hairpin and the N terminus of helix HA of the Tir IBD dimer. The binding interface is primarily hydrophobic, but electrostatic interactions, such as the salt bridge between Lys927 of intimin and Glu312 of the Tir IBD, and backbone interactions also contribute to the specificity and affinity of the interaction. (c) Complementary electrostatics between Tir IBD and intimin. An electrostatic surface potential representation of the Tir IBD-intimin complex as computed by GRASP (Nicholls et al., 1991) is shown here, with red representing negatively charged regions and blue representing positively charged regions. The Tir IBD has an overall negative charge, but the dimerization interface is predominantly hydrophobic. Intimin has a complementary net positive charge with a positively charged tip pointing towards the α-helices of the Tir IBD dimer.

Fiber assembly by the chaperone-usher pathway. A schematic of P pilus assembly is shown. Single letters indicate the corresponding Pap protein. Fiber subunits enter the bacterial periplasm via the Sec system (YEG). In absence of the chaperone, subunits misfold, aggregate, and are proteolytically degraded. The chaperone (PapD) forms a soluble complex with each subunit (including the PapG adhesin, PapF, PapE, PapK, and PapA). The chaperone facilitates the folding of the subunit, stabilizes it, and caps its interactive surfaces while priming it for polymerization. Chaperonesubunit complexes are targeted to the outer membrane usher (PapC), where chaperone dissociation permits subunit incorporation into the growing fiber. The pilus adopts its quaternary structure outside the cell.

10.1128/9781555818395/plate-20_thmb.gif

10.1128/9781555818395/plate-20.gif

Click to view

Color Plate 20 (chapter 5).

Fiber assembly by the chaperone-usher pathway. A schematic of P pilus assembly is shown. Single letters indicate the corresponding Pap protein. Fiber subunits enter the bacterial periplasm via the Sec system (YEG). In absence of the chaperone, subunits misfold, aggregate, and are proteolytically degraded. The chaperone (PapD) forms a soluble complex with each subunit (including the PapG adhesin, PapF, PapE, PapK, and PapA). The chaperone facilitates the folding of the subunit, stabilizes it, and caps its interactive surfaces while priming it for polymerization. Chaperonesubunit complexes are targeted to the outer membrane usher (PapC), where chaperone dissociation permits subunit incorporation into the growing fiber. The pilus adopts its quaternary structure outside the cell.

The chaperone. A ribbon diagram (Carson, 1997) of the crystal structure of PapD is shown. The Nand C-terminal domains, the F1-G1 loop, and the F1 and G1 strands of the chaperone (green) are labeled. The conserved basic residues in the cleft (Arg8 and Lys112) are shown in white (carbon) and blue (nitrogen) ball-and-stick representation. The alternating hydrophobic residues of the F1-G1 loop are in yellow.

10.1128/9781555818395/plate-21_thmb.gif

10.1128/9781555818395/plate-21.gif

Click to view

Color Plate 21 (chapter 5).

The chaperone. A ribbon diagram (Carson, 1997) of the crystal structure of PapD is shown. The Nand C-terminal domains, the F1-G1 loop, and the F1 and G1 strands of the chaperone (green) are labeled. The conserved basic residues in the cleft (Arg8 and Lys112) are shown in white (carbon) and blue (nitrogen) ball-and-stick representation. The alternating hydrophobic residues of the F1-G1 loop are in yellow.

Donor strand complementation. Two representations of the PapD-PapK chaperone-subunit complex are shown. In the upper panel, PapD is in green and PapK is in cyan. The chaperone G1 strand lies between the subunit A2 and F strands and completes its Ig fold. The alternating hydrophobic residues (yellow) of the G1 strand contribute to the hydrophobic core (magenta) of the subunit. The clamping interaction in the chaperone cleft (red, white, and blue ball-and-stick structure) can be appreciated. In the crystal structure, residues 1 to 8 of PapK (the majority of the N-terminal extension) are disordered and not visible. The N-terminal extension label indicates residue 9 of PapK. In the lower panel, PapD is depicted as a worm and PapK is shown in surface representation (Nicholls et al., 1991). The view is looking into the subunit groove. The subunit hydrophobic core is in yellow. The exposed position of the subunit N-terminal extension (residue 9 is indicated) in the complex and the wedging action of the chaperone, which holds the A and F strands away from each other at one end of the groove (the right side of the complex as shown) can be appreciated.

10.1128/9781555818395/plate-22_thmb.gif

10.1128/9781555818395/plate-22.gif

Click to view

Color Plate 22 (chapter 5).

Donor strand complementation. Two representations of the PapD-PapK chaperone-subunit complex are shown. In the upper panel, PapD is in green and PapK is in cyan. The chaperone G1 strand lies between the subunit A2 and F strands and completes its Ig fold. The alternating hydrophobic residues (yellow) of the G1 strand contribute to the hydrophobic core (magenta) of the subunit. The clamping interaction in the chaperone cleft (red, white, and blue ball-and-stick structure) can be appreciated. In the crystal structure, residues 1 to 8 of PapK (the majority of the N-terminal extension) are disordered and not visible. The N-terminal extension label indicates residue 9 of PapK. In the lower panel, PapD is depicted as a worm and PapK is shown in surface representation (Nicholls et al., 1991). The view is looking into the subunit groove. The subunit hydrophobic core is in yellow. The exposed position of the subunit N-terminal extension (residue 9 is indicated) in the complex and the wedging action of the chaperone, which holds the A and F strands away from each other at one end of the groove (the right side of the complex as shown) can be appreciated.

Donor strand exchange. The PapD-PapE chaperone-subunit complex (left; only the G1 strand [green] of the chaperone is shown) and a complex of the PapE subunit (blue) bound to a peptide corresponding to the N-terminal extension (residues 1 to 11) of PapK (red) are shown. Strands are labeled. Note the reversal in orientation and shift in register of the complementing strands on donor strand exchange. Note also the positions of the N and C termini (corresponding to the beginning of the A strand and the end of the F strand, respectively, on either side of the groove) of the subunit in each complex. The displacement of the chaperone allows the A and F strands to move together at this end of the groove, closing it and sealing the N-terminal extension in place. In the chaperone-subunit complex, the loops at the end of the subunit away from its N and C termini are (bottom of view as shown) disordered. The adoption of a more compact, ordered state by the subunit after donor strand exchange can be appreciated.

10.1128/9781555818395/plate-23_thmb.gif

10.1128/9781555818395/plate-23.gif

Click to view

Color Plate 23 (chapter 5).

Donor strand exchange. The PapD-PapE chaperone-subunit complex (left; only the G1 strand [green] of the chaperone is shown) and a complex of the PapE subunit (blue) bound to a peptide corresponding to the N-terminal extension (residues 1 to 11) of PapK (red) are shown. Strands are labeled. Note the reversal in orientation and shift in register of the complementing strands on donor strand exchange. Note also the positions of the N and C termini (corresponding to the beginning of the A strand and the end of the F strand, respectively, on either side of the groove) of the subunit in each complex. The displacement of the chaperone allows the A and F strands to move together at this end of the groove, closing it and sealing the N-terminal extension in place. In the chaperone-subunit complex, the loops at the end of the subunit away from its N and C termini are (bottom of view as shown) disordered. The adoption of a more compact, ordered state by the subunit after donor strand exchange can be appreciated.

Stabilization of the N. gonorrhoeae D-region by hydrophobic packing of conserved β2 Val87, β3 Trp109, and β5 Val125 with β6 Val133 in the hypervariable sequence. Side chain colors, including the yellow disulfide, are the same as in Fig. 2a. For clarity, α1, the αβ loop, β1, and the C-terminal tail (residues 152 to 158) are not included in the view, which is rotated 90° counterclockwise about the vertical axis from its orientation in Fig. 2a.

10.1128/9781555818395/plate-25_thmb.gif

10.1128/9781555818395/plate-25.gif

Click to view

Color Plate 25 (chapter 6).

Stabilization of the N. gonorrhoeae D-region by hydrophobic packing of conserved β2 Val87, β3 Trp109, and β5 Val125 with β6 Val133 in the hypervariable sequence. Side chain colors, including the yellow disulfide, are the same as in Fig. 2a. For clarity, α1, the αβ loop, β1, and the C-terminal tail (residues 152 to 158) are not included in the view, which is rotated 90° counterclockwise about the vertical axis from its orientation in Fig. 2a.

Stereo view of the receptor-binding loop of PAK pilin. Residues 131 to 144 (ball-and-stick representation with main chain atoms in yellow and side chain atoms colored by residue) form a binding surface composed largely of main chain atoms. The disulfide bond (yellow) connects this D-region loop to β4 (green ribbon). Every other side chain is labeled.

10.1128/9781555818395/plate-26_thmb.gif

10.1128/9781555818395/plate-26.gif

Click to view

Color Plate 26 (chapter 6).

Stereo view of the receptor-binding loop of PAK pilin. Residues 131 to 144 (ball-and-stick representation with main chain atoms in yellow and side chain atoms colored by residue) form a binding surface composed largely of main chain atoms. The disulfide bond (yellow) connects this D-region loop to β4 (green ribbon). Every other side chain is labeled.

Type IV pilus filament assembly models. (a) One-start helix with a diameter of 60 Å and a pitch of 41 Å, based on PAK fiber diffraction and used for the N. gonorrhoeae filament model. (b) Surface representation of two turns of the N. gonorrhoeae filament model. The surface is decorated by hypervariable (orange) and semivariable (yellow) residues and posttranslational modifications (red, saccharide; purple, phosphoserine; blue, glycerophosphate [Stimson et al., 1996]), which prevent exposure of invariant (grey) amino acids except in the bottom five subunits. A single subunit is outlined in white. Reprinted from Forest et al. (1999) with permission. (c) Longitudinal filamentpacking interactions of residues 96 to 102 on residues 48 to 56 are supported by endbinding antipeptide antibodies. These two monomers are in the same orientation as the rightmost two in panel b, with the four intervening subunits in the helix removed for clarity. Reprinted from Forest and Tainer (1997b) with permission. (d) Schematic representation of TCP, a 3-start helix with a diameter of 80 Å and a pitch of 45 Å. (Reprinted from Craig et al. (2003) with permission. (e) All-atom surface for 18 subunits of the TCP filament model, representing one complete turn of each strand. Colors match the schematic in panel a, except for a single grey surface to show the boundaries of one monomer. (f) Packing interactions of α3 T122 and α4 Leu176, Ile179, Val182, and Leu185 side chains from the magenta subunit in panel b, with α2 side chains Pro58, Gly72, and Leu76 from the red subunit below it.

10.1128/9781555818395/plate-27_thmb.gif

10.1128/9781555818395/plate-27.gif

Click to view

Color Plate 27 (chapter 6).

Type IV pilus filament assembly models. (a) One-start helix with a diameter of 60 Å and a pitch of 41 Å, based on PAK fiber diffraction and used for the N. gonorrhoeae filament model. (b) Surface representation of two turns of the N. gonorrhoeae filament model. The surface is decorated by hypervariable (orange) and semivariable (yellow) residues and posttranslational modifications (red, saccharide; purple, phosphoserine; blue, glycerophosphate [Stimson et al., 1996]), which prevent exposure of invariant (grey) amino acids except in the bottom five subunits. A single subunit is outlined in white. Reprinted from Forest et al. (1999) with permission. (c) Longitudinal filamentpacking interactions of residues 96 to 102 on residues 48 to 56 are supported by endbinding antipeptide antibodies. These two monomers are in the same orientation as the rightmost two in panel b, with the four intervening subunits in the helix removed for clarity. Reprinted from Forest and Tainer (1997b) with permission. (d) Schematic representation of TCP, a 3-start helix with a diameter of 80 Å and a pitch of 45 Å. (Reprinted from Craig et al. (2003) with permission. (e) All-atom surface for 18 subunits of the TCP filament model, representing one complete turn of each strand. Colors match the schematic in panel a, except for a single grey surface to show the boundaries of one monomer. (f) Packing interactions of α3 T122 and α4 Leu176, Ile179, Val182, and Leu185 side chains from the magenta subunit in panel b, with α2 side chains Pro58, Gly72, and Leu76 from the red subunit below it.

Structure and secretion of Hap. The N-terminal signal sequence is shown in green, the HapS passenger domain is shown in red, the Hapβ translocator domain is shown in dark blue, the putative intramolecular chaperone region at the C terminus of HapS is shown in yellow, and the α-helix (linker) N-terminal to the β-barrel domain is shown in pale blue. The signal sequence targets the preprotein to the inner membrane (IM) and is then cleaved. The C terminus then targets the protein to the outer membrane (OM) and forms a β-barrel with a channel, allowing translocation of the passenger domain to the bacterial surface. Ultimately, the protein undergoes cleavage via an intermolecular event, mediated by the catalytic triad consisting of His98, Asp140, and Ser243, resulting in extracellular release of HapS.

10.1128/9781555818395/plate-28_thmb.gif

10.1128/9781555818395/plate-28.gif

Click to view

Color Plate 28 (chapter 8).

Structure and secretion of Hap. The N-terminal signal sequence is shown in green, the HapS passenger domain is shown in red, the Hapβ translocator domain is shown in dark blue, the putative intramolecular chaperone region at the C terminus of HapS is shown in yellow, and the α-helix (linker) N-terminal to the β-barrel domain is shown in pale blue. The signal sequence targets the preprotein to the inner membrane (IM) and is then cleaved. The C terminus then targets the protein to the outer membrane (OM) and forms a β-barrel with a channel, allowing translocation of the passenger domain to the bacterial surface. Ultimately, the protein undergoes cleavage via an intermolecular event, mediated by the catalytic triad consisting of His98, Asp140, and Ser243, resulting in extracellular release of HapS.

Structure of the translocator domain of the N. meningitidis NalP autotransporter. The β-strands of the NalP β-barrel are shown in shades of blue, and the α-helix that crosses the pore is shown in red (PDB code 1uyn).

10.1128/9781555818395/plate-29_thmb.gif

10.1128/9781555818395/plate-29.gif

Click to view

Color Plate 29 (chapter 8).

Structure of the translocator domain of the N. meningitidis NalP autotransporter. The β-strands of the NalP β-barrel are shown in shades of blue, and the α-helix that crosses the pore is shown in red (PDB code 1uyn).

Models of architecture of Hapβ. (A) Hap_ may reside in the outer membrane as a monomer with a single central pore. (B) Hapβ may form a multimer with a single common pore. (C) Hapβ may form a multimer, with each subunit forming a separate pore.

10.1128/9781555818395/plate-30_thmb.gif

10.1128/9781555818395/plate-30.gif

Click to view

Color Plate 30 (chapter 8).

Models of architecture of Hapβ. (A) Hap_ may reside in the outer membrane as a monomer with a single central pore. (B) Hapβ may form a multimer with a single common pore. (C) Hapβ may form a multimer, with each subunit forming a separate pore.

Structure and secretion of HMW1. The atypical N-terminal signal sequence of HMW1 is shown in green, the secretion domain is shown in pale blue, and the mature protein is shown in red. The HMW1 preproprotein interacts with HMW1C (shown in yellow) in the cytoplasm, resulting in glycosylation (shown as blue circles). Subsequently, the signal sequence targets the preproprotein to the inner membrane (IM) and is then cleaved. The secretion domain then targets the proprotein to the outer membrane (OM), interacting with the HMW1B outer membrane translocator and undergoing cleavage. Ultimately, the mature protein is translocated across the outer membrane and localized on the bacterial surface. Most surface protein is tethered to the bacterial surface, but a small amount is released extracellularly.

10.1128/9781555818395/plate-31_thmb.gif

10.1128/9781555818395/plate-31.gif

Click to view

Color Plate 31 (chapter 8).

Structure and secretion of HMW1. The atypical N-terminal signal sequence of HMW1 is shown in green, the secretion domain is shown in pale blue, and the mature protein is shown in red. The HMW1 preproprotein interacts with HMW1C (shown in yellow) in the cytoplasm, resulting in glycosylation (shown as blue circles). Subsequently, the signal sequence targets the preproprotein to the inner membrane (IM) and is then cleaved. The secretion domain then targets the proprotein to the outer membrane (OM), interacting with the HMW1B outer membrane translocator and undergoing cleavage. Ultimately, the mature protein is translocated across the outer membrane and localized on the bacterial surface. Most surface protein is tethered to the bacterial surface, but a small amount is released extracellularly.

Structure of the secretion domain of B. pertussis FHA. The secretion domain of FHA is a right-handed parallel β-helix (PDB code 1rwr). The regions shown in green and gold are relatively poorly conserved with respect to other TpsA proteins. The region in green serves to cap the N terminus of FHA, and the region in gold extends laterally from the core β-helix. The NPNL and NPNGI motifs are shown in red and form type I β-turns.

10.1128/9781555818395/plate-32_thmb.gif

10.1128/9781555818395/plate-32.gif

Click to view

Color Plate 32 (chapter 8).

Structure of the secretion domain of B. pertussis FHA. The secretion domain of FHA is a right-handed parallel β-helix (PDB code 1rwr). The regions shown in green and gold are relatively poorly conserved with respect to other TpsA proteins. The region in green serves to cap the N terminus of FHA, and the region in gold extends laterally from the core β-helix. The NPNL and NPNGI motifs are shown in red and form type I β-turns.

Structure and secretion of Hia. The atypical N-terminal signal sequence is shown in green; the passenger domain is shown in red, with binding domains in pink; and the translocator domain is shown in dark blue. The signal sequence targets the preprotein to the inner membrane (IM) and is then cleaved. Subsequently, the C terminus forms a trimeric structure in the outer membrane (OM) and facilitates translocation of the passenger domain to the bacterial surface. The functional passenger domain is trimeric.

10.1128/9781555818395/plate-33_thmb.gif

10.1128/9781555818395/plate-33.gif

Click to view

Color Plate 33 (chapter 8).

Structure and secretion of Hia. The atypical N-terminal signal sequence is shown in green; the passenger domain is shown in red, with binding domains in pink; and the translocator domain is shown in dark blue. The signal sequence targets the preprotein to the inner membrane (IM) and is then cleaved. Subsequently, the C terminus forms a trimeric structure in the outer membrane (OM) and facilitates translocation of the passenger domain to the bacterial surface. The functional passenger domain is trimeric.

Molecular features of Hia primary binding domain and YadA collagen binding domain. (A) Structure of HiaBD1 (primary binding domain) and location of the binding pockets on the HiaBD1 trimer surface. (Left) Ribbon diagram of the HiaBD1 monomer structure. Secondary structural elements are labeled from β1 to β13 for β-strands, αA and αB for α-helices, and 310a and 310b for 310-helices, with labels in black, magenta, and green for domains 1, 2, and 3, respectively. (Middle) Ribbon diagram of the HiaBD1 trimer structure. The three subunits are shown in blue, yellow, and red. Only selected secondary-structure elements are labeled. (Right) Electrostatic-potential surface of HiaBD1 trimer. The view represents the same orientation as that in the ribbon diagram of the trimer (middle). The green circles delineate the receptor-binding regions of Hia, containing residues N617, D618, A620, V656, E668, and E678. Note that the entire binding pocket is formed by a single subunit, and thus three identical binding sites are related by a 120° rotation, as illustrated by the solid circle for the red subunit and the dashed circle for the blue subunit. (B) Ribbon diagram of the trimeric YadA collagen-binding domain (PDB code 1p9h). The three subunits are shown in blue, yellow, and red. The trimeric structure is composed of head and neck regions, and the collagen-binding head region adopts a novel nine-coil left-handed parallel β-roll.

10.1128/9781555818395/plate-34_thmb.gif

10.1128/9781555818395/plate-34.gif

Click to view

Color Plate 34 (chapter 8).

Molecular features of Hia primary binding domain and YadA collagen binding domain. (A) Structure of HiaBD1 (primary binding domain) and location of the binding pockets on the HiaBD1 trimer surface. (Left) Ribbon diagram of the HiaBD1 monomer structure. Secondary structural elements are labeled from β1 to β13 for β-strands, αA and αB for α-helices, and 310a and 310b for 310-helices, with labels in black, magenta, and green for domains 1, 2, and 3, respectively. (Middle) Ribbon diagram of the HiaBD1 trimer structure. The three subunits are shown in blue, yellow, and red. Only selected secondary-structure elements are labeled. (Right) Electrostatic-potential surface of HiaBD1 trimer. The view represents the same orientation as that in the ribbon diagram of the trimer (middle). The green circles delineate the receptor-binding regions of Hia, containing residues N617, D618, A620, V656, E668, and E678. Note that the entire binding pocket is formed by a single subunit, and thus three identical binding sites are related by a 120° rotation, as illustrated by the solid circle for the red subunit and the dashed circle for the blue subunit. (B) Ribbon diagram of the trimeric YadA collagen-binding domain (PDB code 1p9h). The three subunits are shown in blue, yellow, and red. The trimeric structure is composed of head and neck regions, and the collagen-binding head region adopts a novel nine-coil left-handed parallel β-roll.

Type III secretion chaperones. (A) Comparison of the SptPSicP structure of Salmonella with the YopE-SycE structure of Yersinia. To create the figure, the chaperones SicP and SycE were aligned from the cocrystal structures with their cognate virulence factors. The molecular surface of SicP is shown, and the SptP and YopE polypeptides are drawn as ribbons in cyan and green, respectively. Two different orientations are shown for clarity. (B) An α-helix of SptP (red with orange side chains) inserts into the hydrophobic groove formed from the highly twisted β-sheet of an outer SicP molecule (aqua with cyan side chains). The hydrophobic groove surrounds the helix, contacting almost all SptP side chains along the length of the helix. The SptP contacts are primarily hydrophobic and are centered on a phenylalanine stacking between residues Phe83 of SptP and Phe36 of SicP. (C) Alignment of four secretion chaperones whose structures have been determined. The hydrophobic groove is marked by an asterisk.

10.1128/9781555818395/plate-35_thmb.gif

10.1128/9781555818395/plate-35.gif

Click to view

Color Plate 35 (chapter 9).

Type III secretion chaperones. (A) Comparison of the SptPSicP structure of Salmonella with the YopE-SycE structure of Yersinia. To create the figure, the chaperones SicP and SycE were aligned from the cocrystal structures with their cognate virulence factors. The molecular surface of SicP is shown, and the SptP and YopE polypeptides are drawn as ribbons in cyan and green, respectively. Two different orientations are shown for clarity. (B) An α-helix of SptP (red with orange side chains) inserts into the hydrophobic groove formed from the highly twisted β-sheet of an outer SicP molecule (aqua with cyan side chains). The hydrophobic groove surrounds the helix, contacting almost all SptP side chains along the length of the helix. The SptP contacts are primarily hydrophobic and are centered on a phenylalanine stacking between residues Phe83 of SptP and Phe36 of SicP. (C) Alignment of four secretion chaperones whose structures have been determined. The hydrophobic groove is marked by an asterisk.

Rho GTPases and bacterial cytoskeletal modulation. (A) Schematic of the complex signaling cascades affected by the Rho GTPases Cdc42, Rac, and Rho. Arrows indicate directions of information flow, and colors roughly segregate the influence of different signaling pathways. This depiction is extremely simplified and is missing many elements, especially of cross talk, in this network. EGF, epidermal growth factor; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid. (B) The regulation of the Rho GTPases at the biochemical level is depicted schematically. The influence of GEFs and GAPs on the nucleotide state is shown, and the function of bacterial toxins (e.g., C3 and CNF) and effectors (e.g., SopE and SptP) is illustrated. (C) The molecular switch of the small GTPases is illustrated by the GTP- and GDP-bound forms of the Ras oncogene. Switch I (yellow) and switch II (red) are shown in their different conformations in each of the structures with different nucleotide. (D) Overview of the full-circle “yin and yang” of host cell manipulation by Salmonella. The type III secretion system is shown to inject virulence proteins (small colored circles) into the host cell (depicted by the thick grey membrane). Actin filaments (black lines) are recruited to the site of bacterial attachment (right side), and actin is polymerized directly by actin-binding proteins (yellow) or indirectly by RhoGTPase activation (by SopE, for example), leading to membrane ruffles (actin-stained cell inset [red] and electron micrograph [blue] with attached bacterium surrounded by ruffles [yellow]). Subsequent shutdown of RhoGTPase signaling (due to SptP) returns the cells to a normal cytoskeletal structure (cell inset with regained actin stress fiber network). At this later stage, Salmonella organisms reside within a complicated vacuole and replicate inside the cell.

10.1128/9781555818395/plate-36_thmb.gif

10.1128/9781555818395/plate-36.gif

Click to view

Color Plate 36 (chapter 9).

Rho GTPases and bacterial cytoskeletal modulation. (A) Schematic of the complex signaling cascades affected by the Rho GTPases Cdc42, Rac, and Rho. Arrows indicate directions of information flow, and colors roughly segregate the influence of different signaling pathways. This depiction is extremely simplified and is missing many elements, especially of cross talk, in this network. EGF, epidermal growth factor; PDGF, platelet-derived growth factor; LPA, lysophosphatidic acid. (B) The regulation of the Rho GTPases at the biochemical level is depicted schematically. The influence of GEFs and GAPs on the nucleotide state is shown, and the function of bacterial toxins (e.g., C3 and CNF) and effectors (e.g., SopE and SptP) is illustrated. (C) The molecular switch of the small GTPases is illustrated by the GTP- and GDP-bound forms of the Ras oncogene. Switch I (yellow) and switch II (red) are shown in their different conformations in each of the structures with different nucleotide. (D) Overview of the full-circle “yin and yang” of host cell manipulation by Salmonella. The type III secretion system is shown to inject virulence proteins (small colored circles) into the host cell (depicted by the thick grey membrane). Actin filaments (black lines) are recruited to the site of bacterial attachment (right side), and actin is polymerized directly by actin-binding proteins (yellow) or indirectly by RhoGTPase activation (by SopE, for example), leading to membrane ruffles (actin-stained cell inset [red] and electron micrograph [blue] with attached bacterium surrounded by ruffles [yellow]). Subsequent shutdown of RhoGTPase signaling (due to SptP) returns the cells to a normal cytoskeletal structure (cell inset with regained actin stress fiber network). At this later stage, Salmonella organisms reside within a complicated vacuole and replicate inside the cell.

Structural basis of bacterial GEF and GAP activities. (A) Structure of the complex of SopE and Cdc42. SopE is shown as a blue polypeptide, and Cdc42 is shown in green. The conserved GAGA loop of SopE (magenta) inserts between switch I (yellow) and switch II (red) of the GTPase, displacing them and impairing their ability to coordinate the nucleotide, which is not bound in the complex. (B) Surface comparison of active Cdc42 and SopE-disrupted Cdc42. The portions of the molecular surface corresponding to switches I and II are in yellow and red, respectively. SopE is shown as a blue ribbon, with the GAGA loop in magenta (residues in the center of SopE are omitted so as not to block the view of the GAGA loop). The switch regions are found in the complex to be in different conformations relative to that in the active form of the GTPase (on the left). (C) The AlF3 transition state complex reveals that the SptP NH2-terminal four-helix bundle GAP domain (blue) inserts a catalytic arginine (shown in white with nitrogens colored blue) into the active site of the GTPase Rac1 (shown in green with the bound GDP nucleotide). The key regulatory and enzymatic elements are also shown, with switch I (orange) and switch II (red), but the tyrosine phosphatase domain of SptP (see Color Plate 38) is omitted for clarity. (D) SptP positions Gln61 of Rac1 through molecular contacts and inserts Arg209 into the active site to stabilize the transition state. Hydrogen bonds are indicated by white dotted lines or as smaller gray dotted lines for weak bonds. AlF3 is shown with the fluorides colored brown and the aluminum colored grey. The magnesium ion and water molecules are shown as large blue and magenta spheres, respectively. GDP carbon bonds are shown in yellow. A large “W” indicates the nucleophilic water molecule positioned by Gln61 of Rac1. The phosphate-binding loops (P-loop) and guanine-binding loops (G-loops) of Rac1 are shown in purple. The bonds proposed to form during the phosphoryl transfer are shown as solid white lines.

10.1128/9781555818395/plate-37_thmb.gif

10.1128/9781555818395/plate-37.gif

Click to view

Color Plate 37 (chapter 9).

Structural basis of bacterial GEF and GAP activities. (A) Structure of the complex of SopE and Cdc42. SopE is shown as a blue polypeptide, and Cdc42 is shown in green. The conserved GAGA loop of SopE (magenta) inserts between switch I (yellow) and switch II (red) of the GTPase, displacing them and impairing their ability to coordinate the nucleotide, which is not bound in the complex. (B) Surface comparison of active Cdc42 and SopE-disrupted Cdc42. The portions of the molecular surface corresponding to switches I and II are in yellow and red, respectively. SopE is shown as a blue ribbon, with the GAGA loop in magenta (residues in the center of SopE are omitted so as not to block the view of the GAGA loop). The switch regions are found in the complex to be in different conformations relative to that in the active form of the GTPase (on the left). (C) The AlF3 transition state complex reveals that the SptP NH2-terminal four-helix bundle GAP domain (blue) inserts a catalytic arginine (shown in white with nitrogens colored blue) into the active site of the GTPase Rac1 (shown in green with the bound GDP nucleotide). The key regulatory and enzymatic elements are also shown, with switch I (orange) and switch II (red), but the tyrosine phosphatase domain of SptP (see Color Plate 38) is omitted for clarity. (D) SptP positions Gln61 of Rac1 through molecular contacts and inserts Arg209 into the active site to stabilize the transition state. Hydrogen bonds are indicated by white dotted lines or as smaller gray dotted lines for weak bonds. AlF3 is shown with the fluorides colored brown and the aluminum colored grey. The magnesium ion and water molecules are shown as large blue and magenta spheres, respectively. GDP carbon bonds are shown in yellow. A large “W” indicates the nucleophilic water molecule positioned by Gln61 of Rac1. The phosphate-binding loops (P-loop) and guanine-binding loops (G-loops) of Rac1 are shown in purple. The bonds proposed to form during the phosphoryl transfer are shown as solid white lines.

Bacterial PTPs. (A) Alignment of the bacterial PTP enzymes (SptP and YopH) together with the eukaryotic PTP1B. The structures align very well, especially in the active site (the key cysteine residue is shown), with SptP differing from the other two by lacking an NH2-terminal helix (on the far left in the image). (B) Representative depiction of the PTP active site. Shown is the SptP active site with coordinated water molecules and an atom of tungstate (phosphate mimic and PTP inhibitor) bound by the key active-site residues, Asp441, Cys481, and Arg487. (C) Comparison of the aligned phosphatase domains of SptP and YopH illustrated with a molecular surface colored by electrostatic potential such that red is negative (acidic) and blue is positive (basic). A “front” and “back” view are given, which are related by the 180° rotation about the axis indicated. (D) Molecular surface representation of the complex of the NH2-terminal domain of YopH (grey) with a phosphotyrosine (red)-containing peptide (blue). The YopH and peptide surfaces are shown partially transparent to reveal the peptide (white) and the main chain of the folded NH2-terminal domain (yellow).

10.1128/9781555818395/plate-38_thmb.gif

10.1128/9781555818395/plate-38.gif

Click to view

Color Plate 38 (chapter 9).

Bacterial PTPs. (A) Alignment of the bacterial PTP enzymes (SptP and YopH) together with the eukaryotic PTP1B. The structures align very well, especially in the active site (the key cysteine residue is shown), with SptP differing from the other two by lacking an NH2-terminal helix (on the far left in the image). (B) Representative depiction of the PTP active site. Shown is the SptP active site with coordinated water molecules and an atom of tungstate (phosphate mimic and PTP inhibitor) bound by the key active-site residues, Asp441, Cys481, and Arg487. (C) Comparison of the aligned phosphatase domains of SptP and YopH illustrated with a molecular surface colored by electrostatic potential such that red is negative (acidic) and blue is positive (basic). A “front” and “back” view are given, which are related by the 180° rotation about the axis indicated. (D) Molecular surface representation of the complex of the NH2-terminal domain of YopH (grey) with a phosphotyrosine (red)-containing peptide (blue). The YopH and peptide surfaces are shown partially transparent to reveal the peptide (white) and the main chain of the folded NH2-terminal domain (yellow).

Structure and function of an actin stapler. (A) The structure of SipA497–669 is shown as a ribbon diagram and reveals that the domain possesses a globular, and not an extended, conformation. The termini visible and modeled in the structure are denoted N and C, respectively, with the corresponding final amino acid number noted in parentheses. Red dots represent peptide at the termini present in the crystals (indicated by the final residue number) but which were not modeled due to disorder. (B) The surface electrostatics of SipA reveal a polarized molecular surface (shown such that blue indicates a basic or positively charged region and red indicates an acidic or negatively charged region). One side of the molecule possesses a large basic patch, whereas the other side is primarily acidic. An actin monomer is shown above SipA, colored by electrostatic potential. (C) Image analysis of electron micrographs of filaments formed from G-actin and SipA497–669 reveals an additional (nonactin) globular mass with extended, nonglobular density. Placed over the actin density is a model of the F-actin filament (red) generated from actin monomers (for clarity, certain portions of the actin structures were omitted). The SipA497–669 crystal structure (yellow) is modeled into the globular core of the nonactin density. In the enlargement of the SipA density in the maps, the N and C termini of the modeled crystal structure are located near the arm density and labeled. (D) The actin filament is shown as two entwined polymers (one in red, the other in cyan) of actin monomers (shown as spheres and labeled) . SipA is drawn as a smaller yellow sphere with two extended arms projecting from opposite sides of the molecule such that they contact actin protomers in opposing strands, as observed in the EM densities.

10.1128/9781555818395/plate-39_thmb.gif

10.1128/9781555818395/plate-39.gif

Click to view

Color Plate 39 (chapter 9).

Structure and function of an actin stapler. (A) The structure of SipA497–669 is shown as a ribbon diagram and reveals that the domain possesses a globular, and not an extended, conformation. The termini visible and modeled in the structure are denoted N and C, respectively, with the corresponding final amino acid number noted in parentheses. Red dots represent peptide at the termini present in the crystals (indicated by the final residue number) but which were not modeled due to disorder. (B) The surface electrostatics of SipA reveal a polarized molecular surface (shown such that blue indicates a basic or positively charged region and red indicates an acidic or negatively charged region). One side of the molecule possesses a large basic patch, whereas the other side is primarily acidic. An actin monomer is shown above SipA, colored by electrostatic potential. (C) Image analysis of electron micrographs of filaments formed from G-actin and SipA497–669 reveals an additional (nonactin) globular mass with extended, nonglobular density. Placed over the actin density is a model of the F-actin filament (red) generated from actin monomers (for clarity, certain portions of the actin structures were omitted). The SipA497–669 crystal structure (yellow) is modeled into the globular core of the nonactin density. In the enlargement of the SipA density in the maps, the N and C termini of the modeled crystal structure are located near the arm density and labeled. (D) The actin filament is shown as two entwined polymers (one in red, the other in cyan) of actin monomers (shown as spheres and labeled) . SipA is drawn as a smaller yellow sphere with two extended arms projecting from opposite sides of the molecule such that they contact actin protomers in opposing strands, as observed in the EM densities.

The acidic cradle of the LRR-containing YopM. (A) Ribbon diagram showing the structure of the LRR of YopM. The two helices capping the repeats are shown in red, and the inner, concave face points up. (B) The surface electrostatics of YopM (oriented identically to the image in panel A) show that the relative charge distribution makes the inner surface more acidic than the outer surface, particularly the ends. The concave inner surface of LRR-containing proteins is thought to represent the binding site for other proteins.

10.1128/9781555818395/plate-40_thmb.gif

10.1128/9781555818395/plate-40.gif

Click to view

Color Plate 40 (chapter 9).

The acidic cradle of the LRR-containing YopM. (A) Ribbon diagram showing the structure of the LRR of YopM. The two helices capping the repeats are shown in red, and the inner, concave face points up. (B) The surface electrostatics of YopM (oriented identically to the image in panel A) show that the relative charge distribution makes the inner surface more acidic than the outer surface, particularly the ends. The concave inner surface of LRR-containing proteins is thought to represent the binding site for other proteins.

Structural mimicry in bacterial virulence. (A) A comparison of the SptP GAP domain and host GAP enzymes reveals that they engage the GTPase in a similar manner but that SptP is differentiated by its smaller size and unique fold. The GTPases are shown as molecular surfaces colored gray, except for the switch I (orange) and switch II (red) regulatory domains, and the nucleotide GDP (green). In the lower righthand corner of this panel, an alignment of the GTPase-bacterial GAP structures is shown, with an emphasis on the catalytic arginine and the protein scaffolding that presents this residue to the GTPase active site. The nucleotide and AlF3 moieties are also shown. (B) Invasin, with an outer membrane-anchoring domain (schematically shown in red) and an integrin-binding extracellular domain (structure shown as a ribbon diagram colored from blue at the N terminus through the spectrum to red at the C terminus), binds the β1-integrin receptor, links the bacterium to the host, and induces integrin mediated signaling that leads to bacterial internalization. Ig, immunoglobulin. (C) The integrin-binding region of invasin mimics the host integrin ligand fibronectin. At the top are shown the molecular surfaces for these two domains. Shown in red and blue are the locations of integrin contacting aspartic acid and arginine residues, respectively. Below is shown the results of aligning the proteins (invasin in green and fibronectin in purple) based on minimizing the distance between these key integrin-contacting residues. (D) Shown is a superposition of the SopE-CDC42 structure alignment with the Tiam1-Rac1 structure. SopE (blue) inserts the GAGA loop (magenta) in a location where the host factor Tiam1 (cyan) inserts a lysine residue (cyan stick model representation) achieving a similar displacement of the switch I and II regions of the GTPases.

10.1128/9781555818395/plate-41_thmb.gif

10.1128/9781555818395/plate-41.gif

Click to view

Color Plate 41 (chapter 9).

Structural mimicry in bacterial virulence. (A) A comparison of the SptP GAP domain and host GAP enzymes reveals that they engage the GTPase in a similar manner but that SptP is differentiated by its smaller size and unique fold. The GTPases are shown as molecular surfaces colored gray, except for the switch I (orange) and switch II (red) regulatory domains, and the nucleotide GDP (green). In the lower righthand corner of this panel, an alignment of the GTPase-bacterial GAP structures is shown, with an emphasis on the catalytic arginine and the protein scaffolding that presents this residue to the GTPase active site. The nucleotide and AlF3 moieties are also shown. (B) Invasin, with an outer membrane-anchoring domain (schematically shown in red) and an integrin-binding extracellular domain (structure shown as a ribbon diagram colored from blue at the N terminus through the spectrum to red at the C terminus), binds the β1-integrin receptor, links the bacterium to the host, and induces integrin mediated signaling that leads to bacterial internalization. Ig, immunoglobulin. (C) The integrin-binding region of invasin mimics the host integrin ligand fibronectin. At the top are shown the molecular surfaces for these two domains. Shown in red and blue are the locations of integrin contacting aspartic acid and arginine residues, respectively. Below is shown the results of aligning the proteins (invasin in green and fibronectin in purple) based on minimizing the distance between these key integrin-contacting residues. (D) Shown is a superposition of the SopE-CDC42 structure alignment with the Tiam1-Rac1 structure. SopE (blue) inserts the GAGA loop (magenta) in a location where the host factor Tiam1 (cyan) inserts a lysine residue (cyan stick model representation) achieving a similar displacement of the switch I and II regions of the GTPases.

Structure of TraC. Ribbon diagram showing the TraC helical bundle structure. The small helical appendage at the side of the helical core is shown in orange. Marked in red are the positions of three amino acids that were shown to play an important role for function and that may be part of an extended patch on the surface of TraC which may participate in interactions with other proteins.

10.1128/9781555818395/plate-42_thmb.gif

10.1128/9781555818395/plate-42.gif

Click to view

Color Plate 42 (chapter 10).

Structure of TraC. Ribbon diagram showing the TraC helical bundle structure. The small helical appendage at the side of the helical core is shown in orange. Marked in red are the positions of three amino acids that were shown to play an important role for function and that may be part of an extended patch on the surface of TraC which may participate in interactions with other proteins.

Structure of HP0525. (A) Ribbon diagram of the HP0525 monomer. (B) Ribbon diagram of the HP0525 hexamer viewed down the large hole formed by the N-terminal domains. Shown in the periphery of the molecule are polyethylene glycol (PEG) molecules that were components of the crystallization condition and were found to interact specifically with the protein in the crystal. By virtue of this interaction, it has been proposed that HP0525 interacts with the inner membrane by using the molecular surface that interacts with PEG molecules in the crystal (Yeo et al., 2000). (C) Ribbon diagram of HP0525 viewed from the side, illustrating the double-ring structure formed by the stacking of the NTDs and the CTDs. (D) Alternating mode of nucleotide binding in the ATPγS-HP0525 complex. Shown are simulated-annealing electron density maps illustrating the relative binding of ATPγS in molecules A and B of the ATPγS-HP0525 complex. The β-phosphate position of ATPγS in site B is co-occupied by a sulfate ion. (E) Structural superposition of unliganded HP0525 (violet) and the ADP-HP0525 complex (gray). The notation A to F is arbitrary, with molecule A defined as the first subunit listed in the PDB coordinate files of the unbound and ATPγS-bound structures. The two hexameric assemblies were overlaid with respect to molecule A of ADP-HP0525. Molecule F exhibits the largest conformational change and is colored in green (apo-HP0525) and red (ADPHP0525), respectively. (F) Structural variability of the NTDs in unliganded HP0525. The overlay was carried out with respect to the CTD of subunit A of ADP-HP0525 (red). Subunit F of apo-HP0525 (green) exhibits the largest rotation (~15° more open) about the hinge region between the NTD and CTD. The coloring scheme for all other subunits is as follows: apo-HP0525_A, dark gray; apo-HP0525_B, pink; apo-HP0525_C, orange; apo- HP0525_D, yellow; apo-HP0525_E, cyan. (G) Model for the mode of action of VirB11 ATPases. The N-terminal and C-terminal domains are represented in pink and light blue, respectively. NTDs locked in a rigid conformation by the binding of ATP and ADP are shown in cyan and yellow, respectively.

10.1128/9781555818395/plate-43a_thmb.gif

10.1128/9781555818395/plate-43a.gif

Click to view

Color Plate 43 (chapter 10).

Structure of HP0525. (A) Ribbon diagram of the HP0525 monomer. (B) Ribbon diagram of the HP0525 hexamer viewed down the large hole formed by the N-terminal domains. Shown in the periphery of the molecule are polyethylene glycol (PEG) molecules that were components of the crystallization condition and were found to interact specifically with the protein in the crystal. By virtue of this interaction, it has been proposed that HP0525 interacts with the inner membrane by using the molecular surface that interacts with PEG molecules in the crystal (Yeo et al., 2000). (C) Ribbon diagram of HP0525 viewed from the side, illustrating the double-ring structure formed by the stacking of the NTDs and the CTDs. (D) Alternating mode of nucleotide binding in the ATPγS-HP0525 complex. Shown are simulated-annealing electron density maps illustrating the relative binding of ATPγS in molecules A and B of the ATPγS-HP0525 complex. The β-phosphate position of ATPγS in site B is co-occupied by a sulfate ion. (E) Structural superposition of unliganded HP0525 (violet) and the ADP-HP0525 complex (gray). The notation A to F is arbitrary, with molecule A defined as the first subunit listed in the PDB coordinate files of the unbound and ATPγS-bound structures. The two hexameric assemblies were overlaid with respect to molecule A of ADP-HP0525. Molecule F exhibits the largest conformational change and is colored in green (apo-HP0525) and red (ADPHP0525), respectively. (F) Structural variability of the NTDs in unliganded HP0525. The overlay was carried out with respect to the CTD of subunit A of ADP-HP0525 (red). Subunit F of apo-HP0525 (green) exhibits the largest rotation (~15° more open) about the hinge region between the NTD and CTD. The coloring scheme for all other subunits is as follows: apo-HP0525_A, dark gray; apo-HP0525_B, pink; apo-HP0525_C, orange; apo- HP0525_D, yellow; apo-HP0525_E, cyan. (G) Model for the mode of action of VirB11 ATPases. The N-terminal and C-terminal domains are represented in pink and light blue, respectively. NTDs locked in a rigid conformation by the binding of ATP and ADP are shown in cyan and yellow, respectively.

Structure of TrwB. (A) Ribbon diagram of the TrwB monomer and domain organization. (B) Ribbon diagram of the TrwB hexamer viewed through the 22-Å mouth of the structure that is expected to face the bacterial membrane. (C) Side view of the sphere-shaped TrwB hexamer and relative positioning to the bacterial membrane.

10.1128/9781555818395/plate-44_thmb.gif

10.1128/9781555818395/plate-44.gif

Click to view

Color Plate 44 (chapter 10).

Structure of TrwB. (A) Ribbon diagram of the TrwB monomer and domain organization. (B) Ribbon diagram of the TrwB hexamer viewed through the 22-Å mouth of the structure that is expected to face the bacterial membrane. (C) Side view of the sphere-shaped TrwB hexamer and relative positioning to the bacterial membrane.

Structure of the relaxases TraI (F) and TrwC (R388). (a) Ribbon diagrams of F-factor TraI and R388 TrwC relaxases (PDB entries 1P4D and 1OMH, respectively). The structures are presented in the same orientation for comparison purposes. Positions of catalytic residues Tyr18 (TrwC) and Tyr16-Tyr23 (TraI) are indicated. Disordered regions of the crystals that could not be traced are depicted as dashed lines. (b and c) Molecular surface diagrams of the crystallographically determined DNA binding cleft in R388 TrwC (b) and localization of residues important for DNA recognition in F factor TraI (c). Also shown are the catalytic residues Tyr18 (TrwC) and Tyr16 and Tyr23 (TraI) in magenta and the putative general bases Asp85 (TrwC) and Asp81 (TraI) in blue. The 25-mer DNA in complex with TrwC is marked to indicate the beginning and end of the sequence at G1 and T25, respectively. DNA segments G3 to C7 and G12 to C16 in the double- stranded hairpin are involved in Watson-Crick base-pairing. Also indicated are the region of the active site of TrwC (dotted circle) and the nic site immediately downstream of T25.

10.1128/9781555818395/plate-45_thmb.gif

10.1128/9781555818395/plate-45.gif

Click to view

Color Plate 45 (chapter 10).

Structure of the relaxases TraI (F) and TrwC (R388). (a) Ribbon diagrams of F-factor TraI and R388 TrwC relaxases (PDB entries 1P4D and 1OMH, respectively). The structures are presented in the same orientation for comparison purposes. Positions of catalytic residues Tyr18 (TrwC) and Tyr16-Tyr23 (TraI) are indicated. Disordered regions of the crystals that could not be traced are depicted as dashed lines. (b and c) Molecular surface diagrams of the crystallographically determined DNA binding cleft in R388 TrwC (b) and localization of residues important for DNA recognition in F factor TraI (c). Also shown are the catalytic residues Tyr18 (TrwC) and Tyr16 and Tyr23 (TraI) in magenta and the putative general bases Asp85 (TrwC) and Asp81 (TraI) in blue. The 25-mer DNA in complex with TrwC is marked to indicate the beginning and end of the sequence at G1 and T25, respectively. DNA segments G3 to C7 and G12 to C16 in the double- stranded hairpin are involved in Watson-Crick base-pairing. Also indicated are the region of the active site of TrwC (dotted circle) and the nic site immediately downstream of T25.

Architecture of the type IV secretion machinery. A model view of the type IV secretion apparatus composed of energizers (red), core complex components (blue), peptidoglycanases (grey), and surface-exposed components (yellow) is shown. The subunits are named according to the VirB-VirD4 secretion system of A. tumefaciens. Substrates secreted into host cells are exemplified by the A. tumefaciens T4SS substrates (green), one of which interacts with a nonsecreted chaperone (VirE1 [white]). IM and OM, inner membrane and outer membrane; the peptidoglycan is schematically represented at the periplasmic/outer membrane surface. Further explanations are given in the text.

10.1128/9781555818395/plate-46_thmb.gif

10.1128/9781555818395/plate-46.gif

Click to view

Color Plate 46 (chapter 10).

Architecture of the type IV secretion machinery. A model view of the type IV secretion apparatus composed of energizers (red), core complex components (blue), peptidoglycanases (grey), and surface-exposed components (yellow) is shown. The subunits are named according to the VirB-VirD4 secretion system of A. tumefaciens. Substrates secreted into host cells are exemplified by the A. tumefaciens T4SS substrates (green), one of which interacts with a nonsecreted chaperone (VirE1 [white]). IM and OM, inner membrane and outer membrane; the peptidoglycan is schematically represented at the periplasmic/outer membrane surface. Further explanations are given in the text.

Crystal structure of PFO. Shown are ribbon representations of the crystal structure of soluble PFO. (A) D1 to D4 are shown in magenta, green, blue, and orange, respectively. The location of the undecapeptide is in white. (B) Shown in red are the α-helical regions of D3 that form the two transmembrane β-hairpins (TMH1 and TMH2). Shown as space-filled atoms are the residues of D4 that anchor PFO to the membrane. The space-filled atoms colored in magenta are the tryptophans of the undecapeptide, and those in yellow are residues of the other hydrophobic loops in D4 shown to penetrate the membrane surface (Ramachandran et al., 2002). The representations in panels A and B were generated using PyMOL (DeLano, 2002).

10.1128/9781555818395/plate-47_thmb.gif

10.1128/9781555818395/plate-47.gif

Click to view

Color Plate 47 (chapter 11).

Crystal structure of PFO. Shown are ribbon representations of the crystal structure of soluble PFO. (A) D1 to D4 are shown in magenta, green, blue, and orange, respectively. The location of the undecapeptide is in white. (B) Shown in red are the α-helical regions of D3 that form the two transmembrane β-hairpins (TMH1 and TMH2). Shown as space-filled atoms are the residues of D4 that anchor PFO to the membrane. The space-filled atoms colored in magenta are the tryptophans of the undecapeptide, and those in yellow are residues of the other hydrophobic loops in D4 shown to penetrate the membrane surface (Ramachandran et al., 2002). The representations in panels A and B were generated using PyMOL (DeLano, 2002).

Proposed model for the cytolytic mechanism of the CDCs. Shown is a proposed model of the binding and membrane insertion of a CDC based on the PFO crystal structure. In panels B to E, only D1, D3, and D4 are visible; D2 is located behind D3. The two transmembrane hairpins are shown in magenta (TMH1) and green (TMH2). (A) Membrane recognition and binding via D4; (B) assembly of the prepore oligomer (only six monomers are shown; a complete oligomer would be composed of 35 to 50 monomers in a circular complex); (C) movement of D3 away from the D2 backbone (directly behind D3) and rearrangement of the secondary structures of the D3 α-helices (in magenta and green) that ultimately form the two transmembrane β-hairpins; (D) proposed formation of a preinsertion transmembrane β-sheet; (E) insertion of the β-sheet to form the transmembrane β-barrel. The twist in the core β-sheet of D3 (Color Plate 47) probably relaxes, which would allow the two TMHs to move to opposite sides of their positions in the monomer and extend into the two transmembrane α-hairpins.

10.1128/9781555818395/plate-48_thmb.gif

10.1128/9781555818395/plate-48.gif

Click to view

Color Plate 48 (chapter 11).

Proposed model for the cytolytic mechanism of the CDCs. Shown is a proposed model of the binding and membrane insertion of a CDC based on the PFO crystal structure. In panels B to E, only D1, D3, and D4 are visible; D2 is located behind D3. The two transmembrane hairpins are shown in magenta (TMH1) and green (TMH2). (A) Membrane recognition and binding via D4; (B) assembly of the prepore oligomer (only six monomers are shown; a complete oligomer would be composed of 35 to 50 monomers in a circular complex); (C) movement of D3 away from the D2 backbone (directly behind D3) and rearrangement of the secondary structures of the D3 α-helices (in magenta and green) that ultimately form the two transmembrane β-hairpins; (D) proposed formation of a preinsertion transmembrane β-sheet; (E) insertion of the β-sheet to form the transmembrane β-barrel. The twist in the core β-sheet of D3 (Color Plate 47) probably relaxes, which would allow the two TMHs to move to opposite sides of their positions in the monomer and extend into the two transmembrane α-hairpins.

Injectosome model. (Left) Proposed structure of the injectosome and the known and hypothetical intracellular effects of SPN. Although apoptosis and interruption of phagocytosis can be induced by the intact injectosome, the intracellular pathways by which this effect is communicated are not known, although both effects appear to be induced by the presence of SPN in the keratinocyte cytoplasm (Bricker et al., 2002). (Right) Schematic of the type III secretion system (for details, see chapter 9). Although the injectosome is functionally similar to the type III secretion system, the latter probably evolved from the flagellar assembly apparatus and is not related evolutionarily to the injectosome.

10.1128/9781555818395/plate-49_thmb.gif

10.1128/9781555818395/plate-49.gif

Click to view

Color Plate 49 (chapter 11).

Injectosome model. (Left) Proposed structure of the injectosome and the known and hypothetical intracellular effects of SPN. Although apoptosis and interruption of phagocytosis can be induced by the intact injectosome, the intracellular pathways by which this effect is communicated are not known, although both effects appear to be induced by the presence of SPN in the keratinocyte cytoplasm (Bricker et al., 2002). (Right) Schematic of the type III secretion system (for details, see chapter 9). Although the injectosome is functionally similar to the type III secretion system, the latter probably evolved from the flagellar assembly apparatus and is not related evolutionarily to the injectosome.

Structures of TLRs and IL-1Rs. (A) Crystal structure of an LRR protein, the RNase inhibitor (Protein Data Bank entry 2BNH). The _-strands are shown as arrowed ribbons, and the α-helices are shown as coiled ribbons. The structure contains 17 β/α LRR repeats. (B) Chemical structures of the antiviral compounds imiquimod (top) and R-848 (resiquimod) (bottom), which activate TLR7 and TLR8. (C) Crystal structure of the complex between the extracellular domain of IL-1RI (green) and IL-1β (cyan) (Protein Data Bank entry 1ITB). The three Ig domains of the receptor are labeled D1, D2, and D3. The disulfide bridges are shown in purple for carbon atoms, and the bridge linking D1 and D2 is indicated by the arrow. (D) Crystal structure of the complex between the extracellular domain of IL-1RI (green) and IL-1ra (cyan) (Protein Data Bank entry 1IRA), shown in the same orientation for D1 and D2 as that used in panel C. Note the different positions of the D3 domains of the two complexes. (E) Crystal structure of the complex between the DD of Drosophila Pelle (green) and Tube (cyan, except for residues 165 to 173, which are shown in purple) (Protein Data Bank entry 1D2Z). Side chains from the C-terminal tail of Tube make crucial contacts with Pelle, and the structural observations are supported by functional studies.

10.1128/9781555818395/plate-50_thmb.gif

10.1128/9781555818395/plate-50.gif

Click to view

Color Plate 50 (chapter 12).

Structures of TLRs and IL-1Rs. (A) Crystal structure of an LRR protein, the RNase inhibitor (Protein Data Bank entry 2BNH). The _-strands are shown as arrowed ribbons, and the α-helices are shown as coiled ribbons. The structure contains 17 β/α LRR repeats. (B) Chemical structures of the antiviral compounds imiquimod (top) and R-848 (resiquimod) (bottom), which activate TLR7 and TLR8. (C) Crystal structure of the complex between the extracellular domain of IL-1RI (green) and IL-1β (cyan) (Protein Data Bank entry 1ITB). The three Ig domains of the receptor are labeled D1, D2, and D3. The disulfide bridges are shown in purple for carbon atoms, and the bridge linking D1 and D2 is indicated by the arrow. (D) Crystal structure of the complex between the extracellular domain of IL-1RI (green) and IL-1ra (cyan) (Protein Data Bank entry 1IRA), shown in the same orientation for D1 and D2 as that used in panel C. Note the different positions of the D3 domains of the two complexes. (E) Crystal structure of the complex between the DD of Drosophila Pelle (green) and Tube (cyan, except for residues 165 to 173, which are shown in purple) (Protein Data Bank entry 1D2Z). Side chains from the C-terminal tail of Tube make crucial contacts with Pelle, and the structural observations are supported by functional studies.

Amino acid sequence alignment of representative TIR domains. Three different subfamilies of TIR domains are shown: the TLR subfamily (pink block), the IL-1R subfamily (green block), and the adaptor subfamily (blue block). The row labeled S.S. denotes the assignment of secondary-structure elements based on the crystal structure information. Residues in β-strands and α-helices are boxed in cyan and yellow, respectively. Residues in the three conserved motifs among the TIR domains are indicated by blue dots, except for the position of the Lpsd mutation, which is in red. Sequences from plant disease resistance (R) proteins are not shown.

10.1128/9781555818395/plate-51_thmb.gif

10.1128/9781555818395/plate-51.gif

Click to view

Color Plate 51 (chapter 12).

Amino acid sequence alignment of representative TIR domains. Three different subfamilies of TIR domains are shown: the TLR subfamily (pink block), the IL-1R subfamily (green block), and the adaptor subfamily (blue block). The row labeled S.S. denotes the assignment of secondary-structure elements based on the crystal structure information. Residues in β-strands and α-helices are boxed in cyan and yellow, respectively. Residues in the three conserved motifs among the TIR domains are indicated by blue dots, except for the position of the Lpsd mutation, which is in red. Sequences from plant disease resistance (R) proteins are not shown.

Structures of the TIR domain. (A) Structure of the TIR domain of human TLR2. The side chains of conserved motifs 1 and 3 are shown and labeled YDA and FW. The BB loop corresponds to conserved motif 2. (B) Structure of CheY, viewed in the same orientation. (C) Structural overlap of the TIR domains of human TLR1 (cyan) and TLR2 (yellow). (D) Molecular surface of the TIR domain of human TLR1. The conserved surface patch for the BB loop is colored in magenta, except that the BB7 residue (the Lpsd mutation site) is shown in red. (E) Schematic model of the interactions between the IL-1Rs and TLRs and the TIRcontaining adaptor molecules. Three types of interfaces in the TIR domain signaling complex are indicated: R face, A face, and S face. The asterisk indicates the site of the Lpsd mutation. The receptor is shown as a heterodimer, but it could also be a homodimer or oligomer.

10.1128/9781555818395/plate-52_thmb.gif

10.1128/9781555818395/plate-52.gif

Click to view

Color Plate 52 (chapter 12).

Structures of the TIR domain. (A) Structure of the TIR domain of human TLR2. The side chains of conserved motifs 1 and 3 are shown and labeled YDA and FW. The BB loop corresponds to conserved motif 2. (B) Structure of CheY, viewed in the same orientation. (C) Structural overlap of the TIR domains of human TLR1 (cyan) and TLR2 (yellow). (D) Molecular surface of the TIR domain of human TLR1. The conserved surface patch for the BB loop is colored in magenta, except that the BB7 residue (the Lpsd mutation site) is shown in red. (E) Schematic model of the interactions between the IL-1Rs and TLRs and the TIRcontaining adaptor molecules. Three types of interfaces in the TIR domain signaling complex are indicated: R face, A face, and S face. The asterisk indicates the site of the Lpsd mutation. The receptor is shown as a heterodimer, but it could also be a homodimer or oligomer.